1. Field of the Invention
The present invention relates to a novel use of a biologically active protein, more specifically, to a novel use of protein p53 as an inhibitor of hepatitis B virus replication.
2. Description of the Prior Art
Hepatitis B virus(hereinafter referred to as `HBV`), a causative agent of acute/chronic hepatitis, consists of a partially double-stranded 3.2 kb circular DNA and from which, four proteins are synthesized: they are the core, polymerase, surface antigen and X-gene product(see: Ganem, D. and Varmus, H. E., Annu. Rev. Biochem., 56: 651-693 (1987)).
Four promoters with unique functions have been identified in HBV genome. The pregenomic/core promoter directs the synthesis of 3.6 kb mRNA which contains all the genetic information encoded by the virus. This RNA serves as a replication intermediate and as a template for the synthesis of core and polymerase(see: Seeger, C. et al., Science, 232: 477-484(1986); Yuh, C. H. et al., J. Virol., 66:4073-4084(1992)). The S promoter and the pre-S promoter direct the synthesis of 2.1 and 2.4 kb RNAs utilized for the generation of pre-S1, pre-S2, and S proteins. X promoter directs the transcription of 0.9 kb RNA specific for the synthesis of X gene product. Liver-specific and differentiation state-specific utilization of these promoters are regulated by the two enhancer elements, i.e., enhancer I(ENI) and enhancer II(ENII). These enhancers along with HNF-1(hepatocyte nuclear factor-1) binding element are largely responsible for the restricted tropism of HBV to hepatocytes(see: Antonucci, T. K. and Rutter, W. J., J. Virol., 63:579-583(1989)).
The mechanism of HBV replication differs from that of other DNA viruses in that, like retroviruses, the reverse transcription step is involved. Upon infection of the hepatocytes, a partially double-stranded genome is converted to a complete double-stranded circular, supercoiled DNA. Employing this as a template, 3.6 kb RNA, which is called the pregenome, is transcribed. The pregenome is packaged into a nucleocapsid and is reverse-transcribed using polymerase as an initiation primer(see: Wang, G. H. and Seeger, C., Cell, 71:663-670(1992); Wang, G. H. and Seeger, C., J. Virol., 67:6507-6512(1993)) to generate the minus-strand, single-stranded DNA. The polymerization of the second strand follows until approximately half of the genome is synthesized, resulting in the generation of partially double-stranded circular genome, which is coated and secreted by the infected cells(see: Tiollais, P. et al., Nature, 317:489-495(1985); Ganem, D. and Varmus, H. E., Annu. Rev. Biochem., 56:651-693(1987)).
Despite extensive epidemiological evidences that chronic carriers of HBV have an increased risk of developing hepatocellular carcinoma(hereinafter referred to as `HCC`) (see: Beasley, R. P. et al., Lancet ii, 1129-1133(1981)), the exact mechanism by which HBV cause HCC still remains unclear. Although integration of HBV DNA into the host chromosome is observed in cancer patients, integration appears to occur at random sites rendering it unlikely that integration-mediated alteration of adjacent gene expression is the cause of HCC(See: Chen, D. S., Nature, 262:369-370 (1993)).
On the other hand, p53, a 53 kDa protein ubiquitous in mammalian cell, was reported to have the function of tumor suppressors. It was first isolated as a coprecipitated form with SV40 large tumor antigen(see: Lane, D. P. and Crawford, L. V., Nature, 278:261-263(1979); Linzer, D. I. H. and Levine, A. J., Cell, 7:43-52(1979)), and its biological function and biochemical properties have been further investigated with the aid of molecular biology, e.g., cDNA cloning of p53 in various organisms. It has been also reported that mutation in p53 tumor suppressor gene is the most frequently occurring event in a wide variety of human tumors(see: Hollstein, M. et al., Science, 253:49-53(1991)). However, the precise mechanism how p53 exerts its tumor suppressing function is not yet clear, except for recent reports suggesting that at least one function of p53 lies in the regulation of transcription.
p53 acts both as a transcription activator and the transcriptional repressor. When working as a transcriptional activator, p53 binds to the enhancer element in a sequence-specific manner(see: Kern, S. E. et al., Science, 252:1707-1711(1991)) and presumably interacts with the basic transcriptional machinery. In case p53 mediates transcriptional repression, p53 forms a complex with the proteins of the basic transcriptional machinery like TATA-box binding protein(see: Seto, E. et al., Proc. Natl. Acad. Sci., USA, 89:12028-12032(1992); Truant, R. et al., J. Biol. Chem., 268:2284-2287(1993); Ragimov, N. et al., Oncogene, 8:1183-1193(1993); Martin, D. W. et al., J. Biol. Chem., 268:13062-13067(1993)) or the CCAAT binding factor(see: Agoff, S. N. et al., Science, 259:84-87(1993)), and inhibits the TATA-box or CCAAT-box-mediated transcription.
It has been reported that p53 is a common target of the transforming proteins of several DNA viruses, e.g., p53 associates with SV40 large T(see: Lane, D. P. and Crawford, L. V., Nature, 278:261-263(1979); Linzer, D. I. H. and Levine, A. J., Cell, 7:43-52(1979)), Adenovirus E1B(see: Sarnow, P. et al., Cell, 28:387-394(1982)), human papilloma virus E6(see: Werness, B. A. et al., Science, 248:76-79(1990)), Ebstein Barr Virus EBNA5(see: Szekely, L. et al., Proc. Natl. Acad. Sci., USA, 90:5455-5459(1993)) and HBV X gene product(see: Feitelson, M. A. et al., Oncogene, 8:1109-1117 (1993)). These viral proteins are thought to abrogate the normal functions of p53 leading to the transformation of infected cells. In SV40 DNA replication, p53 interferes with the ability of T antigen to mediate the viral replication either by binding to replication origin(see: Bargonetti, J. et al., Cell, 65:1083-1091(1991)) or by forming complexes with T antigen(see: Friedman, P. N. et al., Proc. Natl. Acad. Sci., USA, 87:9275-9279(1990)).
Under the circumstances, the present invention have delineated the biological roles of p53 in the replication of HBV, which is a causative agent of frequently occurring acute/chronic hapatitis, liver cirrhosis and HCC.